1. Field of the Invention
The invention relates to the fields of molecular biology and plant genetics. More specifically, the invention relates to plant artificial minichromosome platforms and methods for their production and use.
2. Description of Related Art
Maintenance and inheritance of chromosomes in an organism typically requires three essential elements: origins of replication, a centromere and telomeres, as previously identified with yeast artificial chromosomes. Murray and Szostak (1983) for example described a cloning system based on the in vitro construction of linear yeast artificial chromosomes. However, none of the nucleic acid elements that were identified are sufficient to maintain artificial chromosomes in multicellular eukaryotic systems.
One method for generating an artificial chromosome is by de novo construction, i.e., assembly of centromeres, telomeres and selectable markers and reintroduction into plants (U.S. Pat. No. 7,015,372). However, chromosomes are known to have a minimal size limit for efficient transmission through meiosis (Schubert, 2001). Although most de novo mammalian minichromosomes can transmit during mitosis, the transmission of such minichromosomes in germlines has not been reported. The only meiotically transmitted de novo artificial chromosomes in eukaryotes to date are yeast artificial chromosomes (Murray and Szostak, 1983; Murray and Szostak, 1986). Additionally, it has been indicated that plant centromeres need to be greater than about one megabase in size for normal transmission (Kaszas and Birchler, 1998) and this size is larger than can currently be assembled in vitro.
In contrast, some truncated chromosomes with native centromeres are transmissible through meiosis (Shinohara et al., 2000; Tomizuka et al., 1997, 2000; Voet et al., 2001; Shen et al., 1997, 2000; Schubert, 2001; Zheng et al., 1999; Kato et al., 2005; McClintock, 1938; Brock and Pryor, 1996; Nasuda et al., 2005). One method for truncating chromosomes that has been applied in mammalian systems is telomere mediated truncation (Farr et al., 1991, 1992, 1995; Barnett et al., 1993; Itzhaki et al., 1992; Heller et al., 1996; Mills et al., 1999; Saffery et al., 2001). However, no method for telomere mediated truncation of plant chromosomes has been described to date.
Other methods of chromosome deletion have been applied to plant systems. For example, altered maize chromosomes have been generated by X-ray or gamma irradiation of maize pollen (McClintock, 1938; Brock and Pryor, 1996) or through a B-9 translocation with duplicated 9S (Zheng et al., 1999; Kato et al., 2005). However, altered chromosomes created by these methods have lacked stable transmission during meiosis or mitosis, do not carry site specific recombination sites, have not enabled efficient expression of heterologous sequences or have been difficult to produce. Thus, there remains a great need in the art for methods of producing and using plant minichromosomes.